The present invention is concerned with a process for the semi-quantitative determination of the presence of high levels of glucose in aqueous fluids and with a particular indicator (chromogen) useful in such determination.
The determination of glucose in body fluids, such as urine or blood, is of importance not only in the case of diabetic patients who must control their sugar input, but it is also important in those situations in which the detection of disease as a public health measure requires the screening of the urine or blood of large numbers of people. Because early diagnosis and continued control are so important in diabetes, a glucose test, to be of greatest value to the physician in his diagnosis and control of the disease, must be conveniently rapid, simple enough to serve the clinician and sensitive enough to reflect meaningful variations in urine or blood glucose.
The use of glucose oxidase, a peroxidatively active substance and a chromogen, which is oxidized upon exposure to hydrogen peroxide in the presence of the peroxidatively active substance, for the detection of glucose in urine is known. The system involves the formation of hydrogen peroxide by the action of glucose oxidase on glucose: ##STR1## and the resultant oxidation of the chromogen (CR) to its oxidized state (CR*) which is visually detectable by a color change: ##STR2##
The test described above can be used in the determination of a series of materials which react with oxygen and an oxidase resulting in the formation of hydrogen peroxide. Thus, the system is useful for the detection of occult blood in various body tissues because of the fact that hemoglobin is such a material. Several different chromogens have been reported as being useful in the determination method under consideration. U.S. Pat. No. 3,012,976 discloses the use of o-tolidine, o-toluidine, p-toluidine, o-phenylenediamine, N,N'-dimethyl-p-phenylene-diamine, N,N'-diethyl-p-phenylenediamine, benzidine, p-anisidine, o-catechol and pyrogallol in an occult blood test of the type under consideration. U.S. Pat. No. 3,335,069 involves a test for uric acid and describes the use of o-anisidine and p-anisidine as chromogens.
Of the chromogens disclosed as being useful, few have been actually used in practice. Benzidine was a preferred chromogen, but due to the discovery that it is a potent carcinogen, it lost favor. The discovery that 3,3',5,5'-tetraalkylbenzidines were not carcinogens and the publication of this discovery, led to the obvious expedient of using one of these compounds as the chromogen in a system of the type described above. Such is disclosed in British Patent Specification No. 1,464,359. Page 5 of this specification discloses the observed results with o-tolidine, tetramethylbenzidine and tetraethylbenzidine as chromogen at 0, 50, 100, 250, 500 and 1,000 milligram (mg.) % glucose in the fluid being tested. Each of these materials turns from yellow to bright green when the concentration of glucose increases from 0 to 50 mg. %. As the concentration of glucose increases the color of the oxidized chromogen darkens so that the observed colors were olive-black, black and deep green, respectively. This observation highlights a problem with the semi-quantitative determination of glucose in aqueous fluids because at higher concentrations, known chromogens appear black or very dark green thereby limiting their utility as chromogens in semi-quantitative test devices. Semi-quantitative determination of glucose in urine when the glucose concentration is high, i.e., from about 2,000 to 5,000 mg. % is especially important because urine glucose concentrations in diabetic patients can be as high as 5% or higher. The quantitative estimation of urine glucose to concentrations of 5% is important for at least two reasons: (a) High urine glucose concentrations are likely to be associated with diabetic coma. In emergency situations, it is important to determine whether a state of unconsciousness is diabetic coma. A stat test of this description, indicating a high urine glucose concentration would therefore suggest diabetic coma, (b) Urine glucose levels become elevated if an insufficient amount of insulin has been administered. A test which can estimate higher urine glucose concentrations therefore has greater utility in the therapeutic monitoring of insulin requirements.
The use of m-anisidine as a chromogen in reactions involving peroxides and peroxidase is not reported in the literature. Two references report the use of m-anisidine in systems containing peroxide and trace amounts of Cu.sup.++. They are:
Dolmanova, I. F., et al, "Mechanism of the Catalytic Action of Copper in the Oxidation of a Series of Organic Compounds by Hydrogen Peroxide." Vestn. Mosk, Univ., Khim 1970, 11 (5), 573. PA1 Krause, F., "Organic Inhibitors Which are Converted Into Active Redox Catalysts by Trace Amounts of Cu.sup.++." Oesterr. Chem. Ztg. 68 (2), 54 (1967).
It would be desirable, and it is an object of the present invention to provide an improved method for the detection of glucose in aqueous fluids by the glucose oxidase/peroxidatively active substance system, which method is useful for the semi-quantitative determination of glucose at concentrations of 2000 to 5000 mg. %.